Staphylococcus aureus (MRSA), Since 1961, has emerged as the major general antimicrobial resistant bacteria (AMR) worldwide. MRSA can easily transmit across the hospital system and has mostly gained resistance to medications called beta-lactamases. This enzyme destroys the cell wall of beta-lactam antibiotics resulting in resistance against that respective antibiotic. Daptomycin, linezolid and vancomycin were previously used to treat MRSA infections. However, due to mutations and Single nucleotide polymorphisms (SNPs) in Open reading frames (ORFs) and SCCmec machinery of respective antibody, MRSA developed resistance against those antibiotics. The MRSA strains , when their pan-genomes were analyzed were found the genes involved in invoking resistance against the antibiotics as well as the epidemiology of that respective strain. PENC (penicillin plus potassium clavulanate) is the new antibiotic showing potential in treatment of MRSA though it is itself resistant against penicillin alone. In this review, our main focus is on mechanism of development of AMR in MRSA, how different ORFs are involved in evoking resistance in MRSA and what is the core-genome of different antimicrobial resistant MRSA.In the medical community, antibiotics are revered as a miracle because they stop diseases brought on by pathogenic bacteria. Antibiotics have become the cornerstone of contemporary medical advancements ever since penicillin was discovered. Antibiotic resistance developed among germs quickly, placing a strain in the medical field. Methicillin-resistant  Before discovery of antibiotics, the mortality and lethality brought on by infectious bacteria was significant . AlexandEscherichia coli first showed signs of antibiotic resistance (AR) to penicillin in 1940, indicating that the warning was accurate , followed by 929,000 AR attributed fatalities.According to a recent in-depth analysis published in The Lancet, 204 nations are expected to account for AR-associated deaths (4.95 million) and AR-attributed deaths (1.27 million) in 2019. Western Sub-Saharan Africa has the highest rate of AR-related deaths, with an anticipated 114.8 AR-associated death rate/1lac and 27.3 AR-attributed mortality rate/1lac people. Australia had the lowest fatality rate, with only 28 AR associated mortality per 100,000 people and 6.5 AR-attributed fatalities per 100,000 100,000 people. The six pathogenic germs that will cause the most fatalities in 2019 are listed in the same report . PseudomStaphylococcus aureus, as a prevalent member of body's microbiota, is a Gram + ve bacteria having a spherical morphology. Although this bacteria lives on humans commensally, it can nonetheless be voracious as it can lead to food poisoning and skin infections. Antibiotic-resistant (mainly methicillin-resistant) variant of S. aureus known as MRSA is primarily resistant to beta-lactam antibiotics. MRSA was originally identified in 1961 in the United Kingdom, just a year after methicillin was first employed to cure S. aureus infections  and the Centers for Disease Control and Prevention (CDC), MRSA has been a substantial and grave danger on the virulent bacteria target list, respectively. MRSA al2S. aureus i.e. MRSA is typically resistant to beta-lactam drugs like penicillin (methicillin and oxacillin) and cephalosporin (The antibiotic-resistant (methicillin resistant) variant of losporin . By stoplosporin . By genelosporin . Variouslosporin . Neverthlosporin .2.1Because of its heterogeneity, MRSA exhibits varying levels of anti-β-lactam drug resistance and other components of the culture media in which it is cultured. Only a fewer populations (1 in 106 cfu/ml) shows growth in methicillin concentrations of 5ug/ml or above conc. by showing high resistance levels, whereas the majority of cells of heterogeneous methicillin resistance (HeR) strains (99.9% or above) are susceptible to low concentrations of beta-lactam that are about 1-5ug/mL of methicillin. Homogeneous strains (HoR) can show growth in greater concentrations of methicillin that are around 5ug/mL or above and are resistant to low concentrations of beta-lactam .MRSA's heterogeneity is unstable and subject to alter depending on the environment for growth. When growth conditions are supplied with NaCI or sucrose to provide hypertonicity, or when larger quantities of beta-lactam antibiotic are added, or when the strains are kept at 30 °C in an incubator, HeR strains become homogenous strains (HoR). HoR strains can be transformed into HeR by adding EDTA to the growth medium or by incubating them between 37 and 43 °C. The Agr regulator system's control of gene expression is what leads to the conversion of HeR and HoR under various culture conditions . RepeateIn typical growth conditions, the majority of clinical variants of MRSA develop HeR, and the majority of them exhibit lower to moderate resistance levels, although a small number of subpopulations exhibit high levels of resistance .3meso-diaminopimelic acid). Monobactams, carbapenem, penicillin and cephalosporin are examples of beta-lactam antibiotics. Their beta-lactam rings share structural similarities with the pentapeptide chain's d-Ala-d-Ala. Because of their similarities, anti-beta-lactam drugs get linked to PBP and prevent the cross-binding of glycan stands, which falls under the action of d-Ala-d-Ala substrate. Beta-lactam and binding to PBP results in the aggregation of precursors of peptidoglycan, which in turn triggers hydrolase's autolytic digestion of the old peptidoglycan. This severely compromised the structural integrity of the bacterial cell wall without the formation of fresh peptidoglycan, which causes cell injury from high internal osmotic pressure (By attaching to PBP (penicillin binding protein), that is in charge of GlcNAc (N-acetylglucosamine) and MurNAc (N-acetylmuramic acid) cross-linking, the cell wall of bacteria is affected by beta-lactam antibiotics. A cell wall created by this crosslinking will shield the bacteria from dangers outside the body. Pentapeptide chains typically consists of chains of l-Ala—d-Glu-l-lysine that are linked to MurNAc subunits i.e.-d-Ala-d-Ala  confirmation. Bacteria manufacture the enzyme beta-lactamase to combat the effects of beta-lactam antibiotics. In the periplasmic region, this enzyme hydrolyzes beta-lactam, deactivating it prior to PBP binding . The sen3.1Mec1 and MecRI resemble Bla1 and BlaRI in terms of its morphology, mechanism, functions and molecular organization . Mec1 anPBP2a, a new penicillin-binding protein 2a with a decreased affinity for beta-lactam antibiotics, has taken the place of the PBP that is responsible for the cross-binding of peptidoglycan in MRSA . The mec3.1.1mec genes and SCCmec elements-consisting of 3 distinct ccr (cassette chromosome recombinase) genes i.e. ccrA, ccrB and ccrC, there are 12 allotypes of SCCmec machinery identified so far. At a precise location at the 3′ end of the rlmH gene, each unique ccr gene contains a recombinase that regulates the incorporation and removal of SCCmec elements. It is significant to highlight that antibiotic resistance indicators, which are frequently linked to transposons, appear to accumulate in the J regions of the chromosome. There are sections encircled by IS257 that provides resistance to tetracycline [tet(K)], mercury (merAB), bleomycin (ble), aminoglycosides (aadD), as well as Tn554-like elements that encodes resistance to spectinomycin (spc) and MLS antibiotics [erm(A)] or cadmium (cad), and the last two segments are incorporated duplicates of the RC-replicating plasmids pUB110 and pT181, respectively . Along with methicillin resistance genes, SCCmec types II and III that exhibit multi-resistance also have genes for erythromycin and tetracycline resistance. The community-acquired MRSA strains (CA-MRSA), which are among the virulent infections and infect healthy people outside of hospitals, require SCCmec type IV. Other varieties of SCCmec are uncommon.mecR1 and mecI and the complete or trimmed sets of IS431mec and mecA. As far as our testing goes, mec gene complexes can be divided into four groups based on their structural characteristics:1.Class A: IS431-mecA-mecR1-mecI,2.Class B: IS431-mecA-mecR1-IS1272,3.Class C: IS431-mecA-mecR1-IS431, and4.Class D: IS431-mecA-mecR1.By cloning and analyzing the sequences of SCCmec components from different MRSA strains around the world, we discovered that SCCmec can take on a variety of allotypic forms. There are two crucial genetic components that make up SCCmec. The first component is the mec gene complex, which consists of the regulatory genes ccr gene complex, which consists of the 2 site-specific recombinase genes ccrA and ccrB that are in charge of SCCmec's mobility as well as nearby orfs with unknown functions, is the second crucial area . Additional resistance genes generating bleomycin resistance (ant(4′)), tobramycin, kanamycin, heavy metals and penicillin resistance, and tetracycline resistance are included in the plasmids pUB110, pI258, and pT181 incorporated in SCCmec. Additional resistance gene ermA, which encodes inducible streptogramin, macrolide and lincosamide resistance, is present in the SCCmec-integrated Tn554 (ed Tn554 .4MprF lysinylates phosphatidyl-glycerol (PG) to create the positively charged phospholipid (PL) species, lysyl-PG (L-PG). It has been proposed that the Single nucleotide polymorphisms (SNPs) discovered within the mprF ORFs are connected to a gain-in-function phenotype in terms of daptomycin resistance in MRSA. Daptomycin (DAP) has been used in numerous therapeutic contexts since the FDA approved it in 2003, particularly for resistant MRSA infestations including endocarditis . There hOne of the primary hallmarks of DAP-resistant (DAPr) MRSA variants is gain-in-function mutations in a very small number of genes, mainly in the mprF region . The MprS. aureus (MRSA) strain combinations that were daptomycin-susceptible (DAPs)/daptomycin-resistant (DAPr). It was discovered that the transmembrane tract around the synthase-translocase interface included amino acid changes in 12 out of 22 DAPr isolates, or about 55% of the DAPr strains . Seven out of the twenty-two DAPr isolates (or about 32% of the DAPr strains) had the amino acid sequence changes inside the synthase domain of MprF. Most of the mprF SNPs detected in DAPr strains were found to be localized inside the two MprF loci, based on the locations of the most frequent changes ((i)the central bifunctional domain and(ii)the C-terminal synthase domainThis hinderance was addressed in a study by Bayer and his associates using 22 clinical methicillin-resistant  changes  i.e.(i)tmprF SNPs in DAPr strains with HDP cross-resistance, positive surface charge, and L-PG profiles. Despite the fact that DAPr strains with mprF SNPs in the bifunctional domain had higher resistance to tPMPs than DAPr strains with SNPs in the synthase domain, positive surface charge tests failed to detect this connection. These results demonstrated that both charge-related and charge-unrelated pathways in S. aureus mediate DAP resistance and HDP cross-resistance  (fexA or cfr that encodes a 23SrRNA methylase (The first oxazolidinone class antibiotic to be approved is linezolid (LZD). MRSA and other infectious disorders brought on by Gram-positive bacteria can be effectively treated with this medication. By attaching to 23S rRNA domain V, LZD prevents the assembly of the essential 70S initiation complex that is needed for translation of bacterial proteins, hence inhibiting protein synthesis . There hnd rplC) , procureethylase and pointethylase .S. aureus (LRSA) strains from patient blood cultures  acquired linezolid resistance much more quickly than N315 and that variants that are obtained from the subjects before exposure to linezolid (M5-M7) displayed greater levels of LZD resistance as compared to N315. G2576T mutations were the most prevalent method of linezolid resistance in 40 of 63 (63.5%) LRSA strains . Contrar5.1cfr, which was first discovered in a bovine Mammaliicoccus sciuri strain in 2000 from Germany It's interesting to notice that S. capitis had the highest prevalence of the pLRSA417-like plasmids among the clinical Staphylococcus species isolated from humans. Additionally, the fact that pLRSA417-like vectors were susceptible of interspecies transmission through genetic drift even in vivo was demonstrated by the fact that the homologous plasmids pTZ273 and TZ266 from S. cohnii TZ273 and pTZ266 from S. capitis originated from a single patient. The capability of pLRSA417-like plasmids for conjugative transfer was demonstrated by a study.A clinical MRSA strain from Colombia in 2005 was the first human isolate to have the multi-resistance gene S. aureus RN4220NR and Enterococcus faecalis JH2-2 recipient strains could accept S. aureus plasmids pTZ41, pTZ41, pTZ24, pTZ99, pTZ273, and pTZ273 from human clinical isolates. Additionally, the recipient strain was S. capitis TZ24, which lost the cfr-bearing plasmid in serial passage (named TZ249), and the donor strains were the transconjugants RN4220-cfr and JH2-2-cfr. Nearly 10–3 transconjugants were obtained per receiver, which is a transfer frequency. These findings demonstrated the plasmids' capacity for intraspecies and cross species conjugation as well as S. capitis TZ249′s capacity to ingest pLRSA417-like plasmids .5.2The 23S rRNA genes of MRSA are present in 5 or 6 copies and exhibit numerous intrachromosomal sequence variants . The 23S5.3The table below illustrates the correlation between the minimum inhibitory concentrations of linezolid in variants produced through step by step selection and the copy number of the mutant domain V gene in 23S rRNA. All of the rrn loci in M2/128/15 contained the E. coli numbering type of mutation G2576T, while three of the rrn loci in M3/128/32 had the G2447T type of mutation. As fewer LZD-resistant mutants were chosen step by step from our clinical variants, the number of domain V mutation copies tended to increase. However, only one of the rrn loci in the LZD-resistant mutants created by LZD selection from M13 or N315 strains included the G2236T (MRSA numbering) mutation .5.4The VITEK 2, a computerized susceptibility testing method, undergoes quick determination of minimum inhibitory concentrations (MICs) through the examination of the growing dynamics of bacteria in test cards treated with antibiotics. Standardized interpretive readings of these MICs are provided by the Advanced Experts system (AES). In a nutshell, it consists of a database of MIC concentration for various antibiotic combinations and common resistance mechanisms in many species, together with a number of algorithms. The VITEK 2 isolate's MIC phenotype is compared to every pattern in the database, and the best fit is determined. Users are informed when the estimated framework anticipates clinical resistance to medications to which the bacteria seemed susceptible at breakpoint, and they are also informed when unusual pairings of phenotype and species are noted and warned against  see Tab.Table 2MUtilizing the VITEK 2 technology, the clinical traits of MRSA having the enhanced minimum inhibitory concentrations of LZD in light of the numerous LZD resistance processes have been discovered in MRSA. The presence of the cfr(B), cfr and optrA genes, as well as alterations in the 23S rRNA gene or ribosomal proteins , were all evaluated by using the broth microdilution (BMD) test on 27 of the MRSA variants from fourteen subjects with MICs 8 g/ml of LZD. Four (14.8%) of the 27 variants from 1 subject were found to be LZD-resistant shown by BMD and have the T2500A mutation in 23S rRNA gene. The linezolid-resistant results were significant VITEK 2 mistakes because the remaining 23 were proven to be linezolid sensitive. L3 Gly152Asp was the most often found mutation , and it was exclusively found in isolates that were susceptible to the drug linezolid. No isolates had any L4 or L22 protein changes, optrA, cfr, or cfr(B). The outcomes demonstrate that the L3 Gly152Asp mutation was not significantly associated with LZD resistance, however the 23S rRNA T2500A mutation was. For VITEK 2 LZD-resistant findings, a confirmatory test is indicated due to the possibility of false-positive results .6The incidence of broad antibiotic drug use has increased antimicrobial resistance at the same time. It has been noted that the rise in the incidence of microorganisms that are resistant to antibiotics poses a serious danger to the treatment of infectious diseases. Mobile genetic elements  with genS. aureus. It is attached to the core recognition sites (attB and attSCC), one on the MRSA chromosome and the other on the mec gene machinery itself, to accomplish this. The ORFs of unknown origin (orfX), also known as the universal administration area for SCCmec, has a 15 base pair sequence called attB which is located on the chromosomal end (A gene termed cassette chromosome recombinase (ccr) promotes the site-specific administration of the SCCmec into MRSA genome at a location known as the SCCmec attachment or insertion area. . These romal end .S. aureus (MSSA) changed into MRSA after acquiring the genomic island containing the mecA methicillin resistance determinant  contribution from MRSA strains. The 2,025-gene core genome of S. aureus, which makes up 72% of the whole genome size, is essentially steady, while the number of auxiliary and unique genes  is gradually rising (62 distinct sequence types (STs) of y rising .7.1S. aureus clone MRSA strain USA300 (SSTIs). MRSA infections were widespread in the United States in the 1990 s outside of medical facilities (A prominent source of skin and soft tissue infections in the United States is the cilities . By 2004cilities .S. aureus pathogenicity island 5 (SaPI5), a Panton-Valentine leukocidin-carrying Sa2 prophage, and an arginine catabolic mobile element (ACME) type I element were found (In an early whole-genome sequence (WGS) analysis of a single USA300 strain, a Staphylococcal Cassette Chromosome mec (SCCmec) type IVa element, a re found . These 4re found A referenre found , 3.1 kb re found . The rolre found . In contre found . Core gere found .S. aureus USA300 collection as a whole revealed an evolving pan-genome  predominated in CC8, which was diverse and showed little phylogeographic clustering. The USA300 auxiliary genome was essentially homogenous and made up of components that had previously been connected to this lineage. In more recent samples, SCCmec -ve and ACME -ve USA300 variants started to appear, and the prevalence of the Sa5 prophage increased. Due to enhanced heterogeneity in core genome and temporary alterations in the frequency of specific accessory elements, the analyzed n-genome .7.2S. aureus  398, which can lead to typical MRSA-associated infections in humans, is one of the most significant livestock-associated MRSA genetic lineages. In one study, bioinformatics analysis was used to ascertain the genetic properties of three MRSA CC398 isolates acquired from humans (strains C5621 and C9017) and an animal (strain OR418). Of the three strains, C9017 shown the widest genotype of resistance, including resistance to the antimicrobial classes of aminoglycoside, fluoroquinolone, clindamycin, macrolide and tiamulin. The immune evasion cluster system's scn, sak, and chp genes were only found in OR418. Total 288 specific genes of newly isolated variants were discovered by pangenome analysis, the majority of which are hypothetical or connected to phages. The strongest genetic changes were seen in OR418. In OR418 and C5621, the RNAIII (-hemolysin) gene was without a doubt the most expressed gene, however in C9017, it was not found. The proteome profiles of the several strains revealed significant variations. For instance, OR418 has higher levels of the Sbi (which is an immunoglobulin-binding protein). The findings suggest that the OR418 strain has a high potential for zoonotic transmission since Sbi is a multifunctional immune evasion component in . aureus .7.3Several MRSA lineages, primarily those connected to animals, including CC130, CC49, ST425, CC599, and CC1943, have been identified to contain the mecC-gene. The most frequent mecC lineage is CC130, which is related with ruminants . Due to S. aureus' adaptation to specific host species. A sign for some level of human host adaptability is thought to be the genes of the human specific immune evasion cluster (IEC), in particular. Based on the combination of five genes , this IEC system is found in seven different configurations (types A–G); the scn gene  is included in all IEC types, is frequently used as a marker of IEC-positive isolates and is functionally essential  were detected (associated with phages). SCCmecXI, PBP3, GdpP, and AcrB were identical across all strains in terms of amino acids, however PBP1, PBP2, PBP4, and YjbH proteins showed some variation .7.4VISA (Vancomycin-intermediate Staphylococcus aureus) cells are produced spontaneously by the hVISA isolates through a variety of fascinating processes. In one study, the core genomes of 39 such hVISA strains were analysed. The MRSA linked with the hVISA phenotype's genomic relatedness was not found. In 92% of the hVISA, the alteration Try38 to His in Atl (autolysin) was found. In 11 coding areas with putative roles in virulence, transport systems, carbohydrate metabolism, and tRNA synthesis, we found SNPs and k-mers linked to hVISA. Additionally, genes for lacABCDEFG were downregulated in hVISA whereas genes for capABCDE, sdrD, esaA, esaD, essA, and ssaA were overexpressed. Additionally, whilst arginine, glycine, and betaine were more plentiful in hVISA, valine, threonine, leucine tyrosine, FAD, and NADH were more abundant in VSSA. The purine and pyrimidine route, CoA biosynthesis, amino acid metabolism, and aminoacyl tRNA biosynthesis were among the altered metabolic pathways that was seen in hVISA .Similar results were found in another investigation when a total of 39 MRSA isolates (3.3%) were identified as hVISA (1.4% of MRSA recovered from 2006 to 2008 and 5.6% from 2011 to 2014). 95% of the hVISA strains belonged to clonal complex (CC) 5, which is the largest. Only 6/39 hVISA isolates were classified as such by PAP/AUC, with 6 additional isolates falling within the cut-off range (0.87 to 0.89). (0.9). The Leu-14-Ile (90%) and VraT Glu-156-Gly (90%) amino acid changes in WalK were present in most of the 39 hVISA isolates. Additionally, we discovered 10 alterations in the proteins WalK, VraS, RpoB, and RpoC that are exclusive to hVISA isolates .8Although the molecular mechanisms underpinning the development of CA-MRSA are not entirely understood, during the past 20 years, numerous investigations have observed alterations in the epidemiology of MRSA . IncreasA study is being conducted by Chen and his colleagues to clarify how MRSA's epidemiology is changing. The research led to the discovery of 292 MRSA infections. Sequence type (ST) 5 accounted for the bulk of them (51.4%), followed by ST59 . ST5 MRSA's percentage decreased from 68.3% to 32.1% in 2015, whereas ST59 MRSA's percentage increased from 8.9% to 41.0%. Core-genome phylogenetic analysis revealed that ST59 MRSA isolates had more genotypic diversity than ST5 MRSA isolates in both the healthcare-onset and community-onset categories. According to minimum spanning trees, SRRSH had a cluster of ST5 MRSA infections while most ST59 MRSA infections were scattered. The ST59 MRSA strain, which caused 45.2% of the 93 skin and soft tissue infection cases, exhibited lower rates of levofloxacin (11.8%) and ciprofloxacin (19.1%) resistance than the ST239 and ST5 MRSA isolates. Higher virulence was found in ST59 healthcare-onset MRSA in both the cutaneous infection model and the hemolysis assays. Notably, the levels of pathogenicity of these isolates were comparable to those of common MRSA strains found in the population .8.1Multidrug resistance was common among MRSA isolates as expected, however ST59 MRSA isolates displayed a distinct resistance profile than ST5 and ST239 MRSA . Levoflo9The three medications that are most frequently prescribed to treat MRSA infections are vancomycin, daptomycin, and linezolid . The likIt was discovered that PENC may be effective in treating some MRSA infections. In order to predict medication susceptibility for the PENC phenotype, the MRSA variant’s susceptibility was examined. The disadvantage of using a single component to predict drug susceptibility was highlighted by the discovery that S2-R isolates with susceptible mecA genotypes but PENC-resistant phenotypes expressed at higher levels than S2 MRSA (2.61 vs. 0.98). The data of selected UK-sourced MRSA (n = 74) and MRSA gathered in a previous national survey  were used as the training sets to create a model with accuracy of 0.94 and 0.93 for SRRSH-MRSA and UK-sourced MRSA  validation sets, respectively. The AUROC of this model was 0.96 for SRRSH-MRSA and 0.97 for NAM-MRSA, respectively. Despite the fact that the source of the training set data affects the prediction model's scope of applications, the data demonstrated the efficacy of the machine learning approach in predicting susceptibility from cgMLST findings .10MRSA poses a serious hazard to human health because it can become resistant to any antibiotic to which it is subjected. While antibiotics have only been used clinically for about 80 years, bacteria have long since developed resistance mechanisms. As the usage of antibiotics has increased, bacteria have had to adapt and develop sophisticated ways to live. Different SNPs and mutations in genome of MRSA causes it to develop resistance against long used antibiotics used for MRSA treatment like daptomycin, vancomycin etc. Core and pan-genome analysis of these antibiotic resistant MRSA strains helps scientist to identify the mutations involved in development of resistance and its epidemiology. We expect to be able to treat MRSA infections for the foreseeable future with the help of further development of innovative compounds like PENC, identification of additional therapeutic targets, greater stewardship, and more informed decisions about combination therapy.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


In group A, 37% phosphoric acid was applied after bleaching. In group B, 10% sodium ascorbate was used for ten minutes before 37% phosphoric acid. In group C, 35%3-O-ethyl-l-ascorbic acid plus 50% citric acid solution (35EA/50CA) was applied for 5 min. The subgroups were bonded immediately after bleaching. The SBS was determined with a universal testing machine and analyzed using one-way ANOVA and then Tukey’s HSD tests. Adhesive remnant index (ARI) scores were determined with a stereomicroscope and analyzed with a chi-squared test. The significance level was 0.05. Group C demonstrated significantly higher SBS values than group A (p < 0.001), but was not significantly different than the control group or group C (p > 0.05). The ARI scores were significantly different among the groups (p < 0.001). In conclusion, enamel surface treatment using 35EA/50CA improved the reduced SBS to an acceptable clinical level and reduced the clinical chair time.This study investigates how a new substance, composed of ethyl ascorbic acid and citric acid, affects the shear bond strength (SBS) of metal brackets when bonded to bleached teeth. Forty maxillary premolar teeth were used and randomly placed into four groups ( Hydrogen peroxide is a widely used agent for bleaching teeth . SeveralTo avoid a waiting period, antioxidants have been suggested as a possible solution. One of the most commonly used antioxidant agents in clinical and research studies is sodium ascorbate, which is the sodium salt derived from ascorbic acid. This derivative is a widely used antioxidant to regain the post-bleaching SBS after bleaching without postponing the bonding visit, both in vitro ,13,14,15An ascorbic acid derivative called 3-O-ethyl-l-ascorbic acid, containing an ethyl group at the third carbon position, is extensively used in the cosmetic industry as an antioxidant and anti-aging ingredient ,25. ThisHowever, the effect of the one-step surface treatment for teeth bleaching the SBS between the metallic bracket and bleached enamel has not been evaluated. Thus, the objective of this study is to determine the effect of the one-step surface-treatment reagent that combines citric and 3-O-ethyl-l-ascorbic acids as an antioxidant on the 24 h SBS of metallic brackets bonded to bleached human teeth with a composite resin adhesive. The null hypothesis is that the SBS between the control and bleached groups among different surface-treatment protocols are not significantly different.The Human Research Ethics Committee of the Faculty of Dentistry, Chulalongkorn University approved the study protocol (HREC-DCU 2020-113).The sample size calculation was performed using the G*Power 3.1.9.7 program. The calculation indicated that 28 teeth were required. However, 40 teeth were utilized in the study. The inclusion criteria comprised human maxillary premolars extracted for orthodontic reasons from 17–30-year-old patients with intact buccal enamel surfaces and who had not undergone any chemical treatments, such as peroxide derivatives, acids, bonding agent, vanish, or other types of bleaching. Any teeth with restorations, caries, cracks on the buccal surfaces, hypoplastic areas, fluorosis, or any enamel structure abnormalities were excluded.w/v).A one-step surface-treatment reagent was prepared by mixing 0.35 g of 3-O-ethyl-l-ascorbic acid (EA)  with 0.5 g of citric acid (CA)  in 1 mL of distilled water at 40 °C. The final concentration of the reagent was calculated by dividing the mass of the solute by the final solution volume, assuming that the final solution was approximately 1 mL. Based on this calculation, the approximate concentration of the one-step surface-treatment reagent was 35% for 3-O-ethyl-l-ascorbic acid and 50% for citric acid : 37% phosphoric acid on unbleached teeth for 15 s.Control group (n = 10): 37% phosphoric acid on bleached teeth for 15 s.Group A (n = 10): 10% sodium ascorbate treatment for 10 min on bleached teeth follow by 37% phosphoric acid for 15 s.Group B (n = 10): 35% 3-O-ethyl-l-ascorbic acid plus 50% citric acid solution on bleached teeth for 5 min.Group C , the enamel buccal surfaces were polished using fluoride-free pumice powder with a brush attached to a slow-speed handpiece. The teeth were rinsed and dried using a triple syringe, followed by bleaching.®, Orange, CA, USA). The etched surface was 1 mm wider than the bracket base along every margin. The solution was applied using a disposable applicator and a continuous movement for 15 s and rinsed for 30 s and dried with compressed air (oil-free) for 10 s  was used as the bleaching reagent for the 30 teeth in the remaining subgroups . The bleaching gel was applied as recommended by the manufacturer. The bleaching gel was applied for 4 sessions . Then, the enamel surfaces were rinsed with water (30 s) and air-dried (10 s) (The 35% Hydrogen Peroxide gel (Pola Officed (10 s) .In group B, after bleaching, the teeth’s buccal surfaces were treated using 10% sodium ascorbate with a disposable applicator with a continuous movement for ten minutes, then rinsed in water (30 s), air-dried (10 s), and the tooth’s buccal surface was etched, as described for the control group. In group C, the tooth’s buccal surface was treated with 35EA/50CA under a continuous movement for five minutes, rinsed (thirty sec), and dried (ten sec) with compressed air (oil-free) .® Roth type, TOMY, Fuchu-city, Tokyo, Japan) per the manufacturer’s directions. Each bracket was pressed on the enamel surface using a 100 g force ). To ensure complete resin filling under the bracket base, excess composite was observed at each margin. The excess composite was removed with an amalgam carver. The adhesive was light-cured for 40 s with an Ortho curing light  at an intensity of 2000 mW/cm2. The curing light was calibrated before being used on each group. A radiometer  was used to test the curing light prior to the bonding procedures, and this was repeated twice. A bonding index was used for embedding the tooth in an acrylic block so that the universal machine’s blade and bracket margin were parallel , then TransbondTM PLUS adhesive  was placed on the 0.018″ × 0.025″ slot bracket base  with a load cell of 500 N was used for the SBS assay. The test was performed at a 1 mm/min crosshead speed, in an occluso-gingival direction parallel to the height of the contour. The blade was positioned at the interface between the tooth’s surface and bracket base. The machine recorded the shear force in newtons (Ns). The SBS (MPa) was determined by dividing the shear force by the area of the bracket base. The rectangular shape of the bracket base was measured at its width and height using a digital vernier caliper. To determine the bracket base area, the width and height measurements were multiplied, resulting in a calculated bracket base area of 12.28 mmFollowing the debonding procedure, each tooth’s enamel surface was examined to determine the fracture pattern. The adhesive remnant index (ARI) scores were identified using a stereo microscope  at 20X. The teeth were evaluated by the same observer, and the failures were categorized according to the ARI scores:Score 0: No composite remnants on the enamel surface (failure between the adhesive and enamel).Score 1: Less than 50% of the composite remaining on the enamel surface.Score 2: More than 50% of the composite remaining on the enamel surface.Score 3: The entire composite remained on the enamel surface with an impression of the bracket base on the composite, i.e., adhesive failure.The results were analyzed using the SPSS program . The data’s normality was determined using the Kolmogorov–Smirnov test. One-way analysis of variance (ANOVA) was used for identifying significant differences in SBS among the groups. Tukey’s HSD test was used for post hoc multiple comparisons. The chi-squared test was utilized for analyzing the differences in the ARI scores among the test groups. A significance level of 0.05 was used for all comparisons.p > 0.05). The SBS results are presented in The Kolmogorov–Smirnov test revealed that the data had a normal distribution (p < 0.001) (p < 0.001). Treating the enamel surface with 35% 3-O-ethyl-l-ascorbic acid plus 50% citric acid solutions (group C) significantly increased the shear bond strength compared with the bleached teeth without surface treatment (group A) (p < 0.001). The SBS was not significantly different compared with the control group (p > 0.05) and was not significantly different compared with the surface treatment with 10% sodium ascorbate solution (group B) (p > 0.05). Although applying 10% sodium ascorbate solution followed by 37% phosphoric acid etching (group B) increased the SBS compared with the bleached teeth without surface treatment (group A), the mean value was not significantly different (p > 0.05). The shear bond strength significantly decreased when applying the 10% sodium ascorbate solution followed by the 37% phosphoric acid etching after bleaching for group B compared to the control group (p < 0.001).The one-way ANOVA results indicate that the SBS scores among the groups are significantly different (< 0.001) . Tukey’sThe enamel surface evaluation after bracket debonding revealed that significant differences existed in the ARI scores among the four groups. The ARI scores in the bleaching groups  were typically 0. In contrast, score 2 was most commonly identified in the control-group specimens .Using hydrogen peroxide as a bleaching reagent produces free radicals and reactive oxygen species. Free radicals are any molecules that have one or more unpaired electrons resulting in high reactivity. These molecules can interact with the pigments present in the tooth structures that have a high number of electrons, causing the degradation of larger pigmented molecules into smaller molecules with less pigmentation .The present study investigated the effect of 35% 3-O-ethyl-l-ascorbic acid/50% citric acid, a new reagent for surface treatment, on the SBS of metal brackets bonded to bleached human teeth with a composite resin adhesive. We observed that the SBS was significantly different among the groups. Therefore, the null hypothesis was rejected.Our results reveal that using 35% hydrogen peroxide in office for tooth bleaching significantly decreased the shear bond strength after immediate bonding, which was similar to the previous studies ,15,38. TThe 10% sodium ascorbate pretreatment group demonstrated an increase in immediate SBS compared with the untreated bleached enamel; however, the difference was not significant. Moreover, we observed that using 10% sodium ascorbate as a pretreatment solution significantly lowered the shear bond strength compared with non-bleaching. These results imply that 10% sodium ascorbate pretreatment may not be suitable for restoring the SBS. Our results differ from the findings presented in previous studies ,14,17,20Our results indicate that using a one-step surface-treatment reagent, i.e., 35% 3-O-ethyl-l-ascorbic and 50% citric acids, resulted in a significant increase in immediate SBS compared with the bleaching group without surface treatment. Furthermore, the SBS of the treated group was not significantly different compared with the unbleached control group or the group receiving a 10% sodium ascorbic surface treatment. Our results suggest that the one-step surface-treatment reagent could be a viable option for restoring SBS while reducing the treatment time and steps. The possible mechanism for restoring the SBS is that etching the enamel with a solution containing citric and 3-O-ethyl-l-ascorbic acids effectively neutralizes reactive oxygen species. The reason why the 5 min surface treatment resulted in a good shear bond strength comparable to the control, and not significantly longer than 10 min with the 10% sodium ascorbate solution, was that the reaction reached its maximum value after approximately 1 min, followed by a significant reduction. Our study results align with those of Freire et al., who revealed that the reduction reaction kinetics between sodium ascorbate and hydrogen peroxide were dose-dependent and the antioxidant efficiently neutralized the oxidizing agent in 5 min . FurtherRemoving the resin on the enamel surface after bracket debonding is clinically favorable because it can minimize the damage incurred during debonding. ARI scores are commonly used to assess the bracket debonding interface . The ARIThere are several in vitro studies that used different natural antioxidants, including pine bark extract, green tea extract, grape seed extract, and lycopene, as a surface treatment following tooth bleaching and before the bonding process ,22,23. TOne limitation of this study was that it was performed in vitro, and therefore may not fully reflect the clinical condition. Future research should investigate the in vivo effectiveness of the new reagent because, at present, there is no evidence regarding the survival rate of brackets bonded to bleached teeth that have undergone antioxidant surface treatment. However, SBS can be used as an indicator of the bracket survival rate . AnotherThe clinical application of this study is that bleaching with 35% hydrogen peroxide significantly decreased the shear bond strength, and surface treatment with 10% sodium ascorbate prior to bonding of in-office bleaching with 35% hydrogen peroxide did not significantly improve the shear bond strength. The 35% 3-O-ethyl-l-ascorbic acid/50% citric acid solution can regain the decreased SBS. Our new reagent can be used as an option rather than 10% sodium ascorbate to regain the decreased SBS and reduce the treatment steps and clinical chair time.In conclusion, our study determined that bleaching teeth with 35% hydrogen peroxide for 8 min per round for four rounds significantly reduced the 24 h SBS between metal brackets and human teeth. Furthermore, although 10% sodium ascorbate followed by 37% phosphoric acid treatment increased the 24 h SBS compared with bleaching alone, the difference was not significant. Moreover, surface treatment with a one-step reagent composed of 35% 3-O-ethyl-l-ascorbic acid/50% citric acid for 5 min significantly restored the reduced bond strength compared with the bleaching group and was not significantly different compared with the unbleached teeth. Notably, the one-step surface treatment reagent reduced the treatment steps and clinical chair time compared with the 10% sodium ascorbate followed by 37% phosphoric acid treatment.The adhesive remnant index (ARI) score revealed differences among the groups. The bleaching group without surface treatment had the highest frequency of failure at the tooth surface and adhesive interface, in contrast with the control group, where the majority of failures occurred in the adhesive layer. The surface-treatment groups with 10% sodium ascorbate and 35% 3-O-ethyl-l-ascorbic acid/50% citric acid reagent had a mixed pattern of failure at the interface between the enamel and adhesive and within the adhesive layer.Overall, our results suggest that surface treatment with a one-step reagent composed of 35% 3-O-ethyl-l-ascorbic acid/50% citric acid for 5 min is a promising option for restoring the bond strength of teeth after bleaching and may be a more efficient alternative to traditional treatments, such as 10% sodium ascorbate followed by 37% phosphoric acid. However, further studies are needed to confirm our results and explore the optimal application protocols of the one-step surface-treatment reagent in the clinical setting.


Introduction: In previous studies, the relationship between BMD  and LDH (lumbar disc herniation) has been investigated in young people, except for postmenopausal women. The aim of this study was to evaluate this association in postmenopausal women.Methods: A cross-sectional analysis of 545 consecutive postmenopausal women was performed at a single center. The study included patients aged 45 to 85 with low back pain. Age, weight, height, L1-L4 BMD, L1-L4 T-score, L1-L4 Z-score, femoral neck BMD, femoral neck T-score, and femoral neck Z-score of patients were collected. MRI scans were assessed for the diagnosis of LDH. To explore the impact of the number of herniated segments, patients with LDH were further divided into single-level and multi-level LDH groups.Results: Five hundred and thirteen postmenopausal women were included in the final analysis. The mean age of the patients was 61.3±8.6 years in the LDH group and 58.4±7.8 years in the non-LDH group (p=0.001). The LDH group had higher lumbar (p<0.001) and femoral neck (p<0.05) BMD, T, and Z-scores than the non-LDH group. In binary logistic regression analysis, age, lumbar, and femoral neck BMD were significantly associated with LDH . Patients with multi-level herniation had significantly higher rates of obesity (BMI ≥30) compared to patients with single-level herniation . However, in terms of obesity rates, the LDH group and the non-LDH group were statistically similar . There was no association between the single and multi-level herniation groups in terms of L1-4 and femoral neck BMD .Conclusion: Higher lumbar bone mineral density and higher femoral neck bone mineral densities were found to be associated with lumber disc herniation in postmenopausal women with low back pain. These results suggest that bone mineral density assessment may be useful in clinical practice to determine which patients are at higher risk of lumbar disc herniation. Lumbar disc herniation (LDH) is one of the most frequent diseases that has a serious impact on people's quality of life and puts a heavy burden on families and society . It is aSince bone mineral density (BMD) levels affect not only the structure of bones but also nonosseous tissues, osteopenia and osteoporosis disrupt the morphology of lumbar vertebral bones and intervertebral discs ,9. On thClose contact between the vertebral bone marrow and the vertebral endplate is important for adequate nutrition of the nucleus pulposus of the disc . High BMStudy designThis study was a retrospective cross-sectional analysis based on an electronic database from a single center.Patient selectionAn analysis of 545 consecutive patients at a state hospital between January 2021 and February 2022 was performed. Inclusion criteria were a diagnosis of low back pain with or without radiculopathy that persisted for at least one-month, postmenopausal women, patients aged 45 to 85 years, and patients who had undergone both a BMD and a lumbar MRI within one month. Patients with malignancy, inflammatory joint disease, vertebral fractures, a history of spinal surgery for any reason, and those using medications that could affect bone metabolism were excluded from the study.Outcome measures and confoundersAge, weight, height, L1-L4 BMD, L1-L4 T-score, L1-L4 Z-score, femoral neck BMD, femoral neck T-score, and femoral neck Z-score of the patients were collected from the electronic database of the hospital. Patients' weights and heights were measured before BMD measurements using a Soehnle Professional 7830 electronic scale (100g precision) and a standard stadiometer (1mm precision), respectively. Body mass index (BMI) was calculated by dividing the patient's body weight by the square of their height and was expressed as kg/m². BMI was stratified as: 18.5 to 24.9 kg/m², normal; 25.0 to 29.9 kg/m², overweight; 30.0 to 39.9 kg/m², obese; and ≥40 kg/m², morbidly obese, according to the World Health Organization (WHO) definitions.According to WHO guidelines, a T-score between -1 and -2.5 was diagnosed as osteopenia, and a T-score of -2.5 or less as osteoporosis. Patients with a T-score of -1 or above were included in the normal BMD group. BMD measurements were performed using the dual energy X-ray absorptiometry (DXA) method using the LUNAR DPX densitometer . MRI scans were obtained using a 1.5 Tesla machine . LDH was defined as "localized or focal displacement of disc material beyond the limits of the intervertebral disc space," according to the latest recommendations of the North American Spine Society . The terStatistical analysisData were included in a database created by the Excel 2007 program by Microsoft . Statistical analysis was performed using PASW statistics for Windows . The Shapiro-Wilk test was used to determine whether the continuous variables were normally distributed. Continuous variables that provided the parametric test assumption in comparison between groups were tested with Student's t-test. Categorical variables were compared using the Chi-Square test. A binary logistic regression analysis was performed to adjust for the effects of covariates, including age and BMI. To explore the impact of the number of herniated segments, patients with LDH were further divided into single-level and multi-level LDH groups. An ANOVA test was performed to analyze the relationship between herniated segment numbers and BMD. A p-value of <0.05 was considered significant.Characteristics of patientsFive hundred and forty-five postmenopausal women were initially enrolled in the study. After the exclusion of six patients with malignancy, 12 patients with vertebral fractures, two patients with rheumatoid arthritis, nine patients with a history of spinal surgery, one patient using glucocorticoids, and two patients using anti-osteoporotic drugs, 513 patients were included in the final analysis. Four hundred and six patients had at least one level of LDH, and 107 patients had normal MRI findings. The mean age of the patients was 61.3±8.6 years in the LDH group and 58.4±7.8 years in the non-LDH group (p=0.001). The two groups were comparable in terms of height, body weight, and BMI   Table . In binaThe relationship between lumbar disc herniation and T-scoresThe proportion of patients with a normal L1-L4 T-score was 58.4% in the LDH group and 41.1% in the non-LDH group . The proportions of patients with a normal femoral neck T-score were 59.6% and 48.6% in the LDH group and non-LDH group, respectively  (Table 0-3.10). Analysis of patients according to the number of herniated segmentsThere was no association between the single and multi-level herniation groups in terms of L1-4 and femoral neck BMD .Patients with multi-level herniation had significantly higher rates of obesity (BMI ≥30) compared with single-level herniation . However, in terms of obesity rates, the LDH group and the non-LDH group were statistically similar .While the effect of BMD on disc degeneration and osteoarthritis has been widely observed in previous studies -20, the Lumbar end plates are the main anatomical structures that carry loads from bone to disc. Stronger bones mean more load from end plates to intervertebral discs . These aSeveral studies have reported that osteophytes and end plate sclerosis may cause a false increase in DXA measurements -24. TherThe incidence of LDH has been shown to increase with age ,10,11. IIn a previous study, BMI was significantly higher in patients with ≥2 herniated segments than in healthy adults . In the Consistent with previous literature , the numLDH is one of the most common causes of low back pain . LDH wasDifferently from previous studies regarding LDH, we selected our study group from postmenopausal women. With the rapid decrease in estrogen levels, menopause both causes rapid loss of bone mass as well as accelerated lumbar disc degeneration ,29. As wHigher lumbar bone mineral density and higher femoral neck bone mineral density were found to be associated with lumber disc herniation in postmenopausal women with low back pain. These results suggest that bone mineral density assessment may be useful in clinical practice to determine which patients are at higher risk of lumbar disc herniation. According to this assessment, preventive methods such as activity modification, exercise programs, and frequent radiological follow-up can be recommended to patients before lumbar disc herniation develops or progresses. Considering the retrospective cross-sectional analysis, further longitudinal and prospective studies are needed to confirm the results of the current study.


Senescence is characterized by an aggravated inflammatory state that reduces vaccine responsiveness. Dietary supplementation with spray-dried porcine plasma (SDP) exerts anti-inflammatory effects in different mucosal areas. We aimed to determine if the anti-inflammatory properties of SDP improve the efficiency of immunization in senescent animals.Staphylococcus aureus enterotoxin B (SEB) or vehicle every 15  days . Fifteen days after the last dose, a lethal shock was induced by intraperitoneal administration of SEB and LPS.Experiments were performed in 2-month-old and 6-month-old male SAMP8 mice fed control or SDP (8%) feeds for 4  months. The mice received nasal doses of 2.5  μg of p < 0.05). After the lethal shock, all immunized aged mice that were supplemented with SDP survived, in contrast to only 66% of those fed the control feed (p < 0.05). Moreover, after the lethal challenge, aged mice showed higher expression levels of pro-inflammatory cytokines  in jejunal and  in lung tissues (p < 0.05), which were reduced by SDP supplementation (p < 0.05). Furthermore, in senescent mice, SDP supplementation augmented Il-4 and Il-10 expression in both tissues (p < 0.05).Immunization increased anti-SEB IgA in intestinal and bronchoalveolar fluid (SDP reduces the mucosal inflammation associated with aging, improving vaccine protection in senescent mice. These age-associated immune changes are known as immunosenescence and promote a pro-inflammatory profile called inflammaging . MoreoveS. aureus enterotoxin B (SEB)  , 8. Diet B (SEB) , 10. Fur B (SEB) . The eff B (SEB) , 13 and  B (SEB) .Therefore, the aim of the present work was to assess whether the anti-inflammatory properties of SDP can improve vaccine protection in senescent mice. SAMP8 are mice prone to accelerated senescence and are commonly used in aging studies . Moreove2.2.1.Male mice of the SAMP8 strain (prone to accelerated senescence) were obtained from Envigo . The colony generated was maintained at the animal facility of the Faculty of Pharmacy and Food Science of the University of Barcelona. Animals were housed under constant temperature and humidity conditions, with a 12-h:12-h light:dark cycle. All animal experiments were performed following the Guide for the Care and Use of Laboratory Animals, and the protocols used in this study were approved by the Ethics Committee for Animal Experimentation of the Universitat de Barcelona and the Catalan government . Animals were housed individually and fed experimental feeds (control or SDP) for 4 months, from 2 months of age to 6 months.The second of the two experimental feeds (SDP) comprised feed supplemented with 8% SDP. SDP is a feed ingredient obtained by the centrifugation of blood from healthy pigs, which is then spray-dried to obtain a stable powder. SDP maintains the native structure of its proteins, as confirmed by immunoelectrophoresis and western blotting . Both co2.2.The study was divided into two experiments. The first focused on determining how aging and SDP supplementation affect variables involved in immunization while the second tested whether SDP supplementation modified the efficacy of immunization after a lethal shock.In both experiments, the animals were separated into three groups: 2M, 2-month-old mice (young reference group); 6M, 6-month-old senescent mice that had consumed the control feed; and 6M-SDP, 6-month-old senescent mice that had consumed the SDP feed for the previous 4 months . The fee2.2.1.The three experimental groups  were also divided into immunized (Imm) and non-immunized (Non-Imm) animals. Therefore, the experimental groups in this experiment were: non-immunized young mice (2M), immunized young mice (2M-Imm), non-immunized senescent mice fed control feed (6M), immunized senescent mice fed control feed (6M-Imm), non-immunized senescent mice fed SDP feed (6M-SDP), and immunized senescent mice fed SDP feed (6M-SDP-Imm).To perform the immunization procedure, mice were intranasally administered 2.5 μg of SEB  with 5 μg of cholera toxin  as immune adjuvant. Non-immunized mice received CT alone. All immunized animals received three vaccination doses every 2 weeks, as described previously . The you2.2.2.To evaluate the protective efficiency of immunization in senescent mice and the possible effect of SDP supplementation, mice were challenged with a lethal shock comprising SEB and lipopolysaccharide (LPS) as described previously . Briefly2.3.At the end of the experimental period, mice were anesthetized with xylazine/ketamine. Blood was directly collected from the heart and the mice were euthanized during the intervention. Once obtained, samples of serum and jejunal and lung tissue were quickly frozen at −80°C for further analysis.The intestinal fluid was obtained after the small intestinal lumen was washed with phosphate-buffered saline (PBS). The liquid obtained was clarified by centrifugation and stored at −80°C for further analysis.2.4.Peyer’s patches were obtained as previously described . Immune 2.5.g for 10 min to pellet BALF cells. Supernatants were stored at −80°C for the further measurement of immunoglobulins (Igs) while the pelleted cells were suspended in PBS with 5% fetal calf serum . Immune cell recruitment in BALF was assessed by counting total cells obtained. In all cases, cell viability exceeded 95%.Bronchoalveolar lavage fluid (BALF) was obtained as previously described . BALF su2.6.5 cells, as described previously by Miró et al. . Briefly, 96-well polystyrene plates  were coated with anti-mouse IgA monoclonal antibody  in PBS for quantification of the total IgA concentration or with SEB  in PBS for measurement of the anti-SEB-specific IgA titer. After the plates were washed, samples and standards were added and incubated for 2 h. Mouse IgA  was used as a standard for the total IgA determination and an SEB-specific IgA standard curve was derived from serial dilutions of a pool of SEB-positive samples. Horseradish peroxidase-conjugated goat anti-mouse IgA (Bethyl) was used as the detection antibody.Total IgG and SEB-specific IgG concentrations were determined in serum. As capture antibodies, we used goat anti-mouse IgG  to quantify the total IgG concentration or SEB  in PBS to measure the SEB-specific IgG concentration. After the plates were washed, samples and standards were added and incubated for 2 h. As standard, mouse IgG (Sigma-Aldrich) was used for total IgG determination while SEB antibody  was used to quantitate the SEB-specific IgG concentration. Horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma-Aldrich) was used as the detection antibody.In all cases, o-phenylenediamine  was used as HRP substrate, and the color intensity was measured at 492 nm in a microplate reader .2.8.® Green Supermix (Bio-Rad). The primers used are listed in Gusβ) gene expression as reference and was performed with the 2−ΔΔCT method . The effect of immunization on leukocyte recruitment, B cell percentage, and Ig concentration were analyzed by two-way ANOVA using the factors immunization (Imm vs. Non-Imm) and age-feed group . The effects of immunization and SDP supplementation on body weight and food intake were analyzed by three-way ANOVA with repeated measures, using the factors immunization (Imm vs. Non-Imm), age-feed group , and time. Survival after shock induction was analyzed with a log-rank (Mantel-Cox) test. To evaluate the effects of lethal shock on cytokine expression, we considered the factors immunization (Imm vs. Non-Imm) and age-feed . Differences were considered significant at p < 0.05.Results are presented as mean ± SEM. All data were analyzed with the Levene test to assess homogeneity of variance and with the Shapiro–Wilk test to assess distribution. Homogeneous and normally distributed data were analyzed with parametric two-way analysis of variance (ANOVA) or with three-way ANOVA followed by Fisher’s least significant difference (LSD) 3.3.1.p < 0.001), without changing food intake.Immunization with SEB did not modify food intake or changes over time in body weight . On the 3.2.3.2.1.p < 0.001, p = 0.035) because immunization reduced cell recruitment in young mice and had no effect on aged mice. Senescence also reduced the percentage of B lymphocytes in Peyer’s patches  while immunization did not modify it. The percentages of B and IgA+ B lymphocytes in BALF were not modified by aging, feed, or immunization . Immunization with SEB increased the concentration of SEB-specific IgA in the luminal content (p = 0.002).Aged mice had a higher concentration of total IgA in the intestinal lumen compared with young mice . Immunization did not modify this variable but greatly increased the concentration of SEB-specific IgA in BALF . The titer of SEB-specific IgG in serum was similar among experimental groups and increased after immunization in all groups . Immunization also protected senescent mice from the shock .All groups received a lethal shock 3.3.2.p < 0.05, Il-6, Il-1β, Tnf-α, and Ifn-γ in the jejunal mucosa compared with young mice . Senescent mice fed the SDP-supplemented feed had lower expression of all of them . Furthermore, immunization showed a greater protective effect on Il-6 and Ifn-γ expression in animals supplemented with SDP (both p for interaction <0.05).Immunization protected mice from excessive pro-inflammatory cytokine production in the intestinal mucosa during lethal shock in all groups . Senescent mice receiving the SDP-supplemented feed exhibited lower expression of them in response to lethal shock than animals receiving the control feed (both p < 0.05).With respect to lung tissue, immunization protected mice from the excessive production of pro-inflammatory cytokines in response to lethal shock in all groups .Immunization reduced the expression of 4.Older people have a persistent low-intensity pro-inflammatory profile called inflammaging , which iS. aureus in the intestinal microbiota, because dysbiosis associated with aging leads to higher levels of pathogenic and opportunistic microorganisms  in young pigs . In addiIn conclusion, in this study, we have established a model of nasal immunization in senescent mice that protected them mucosally and systemically, as reflected by increased levels of anti-SEB-specific Igs and a higher survival rate to lethal shock with SEB. Furthermore, dietary supplementation with SDP reduced the basal inflammation associated with aging, improving the efficacy of immunization in terms of the response to the lethal shock. The results of this study, combined with previous research, suggest that SDP can enhance the protection of vaccination by reducing the pro-inflammatory state associated with senescence. This finding opens up the possibility of using SDP to improve immune protection in the elderly population. However, it should be noted that this study was conducted only in males, and further research is needed to understand the level of protection provided by SDP in females, as well as the underlying mechanisms before it can be applied to humans. Nonetheless, given that SDP is already being used in farm animals, its potential use in domestic animals appears to be more feasible.The raw data supporting the conclusions of this article will be made available by the corresponding author, without undue reservation.The animal study was reviewed and approved by Ethics Committee for Animal Experimentation of the Universitat de Barcelona and the Catalan government .LM, CA, JP, MM, and AP-B conceived and design the experiments. LM, CR-C, and AP-B conducted research and data analysis. JP contributed with essential reagents and materials. LM, CR-C, and AP-B wrote the original draft preparation. CA, JP, and MM review and edited the manuscript. All authors contributed to the article and approved the submitted version.This research was funded by APC-Europe S.L.U.  by research contracts with the Bosch i Gimpera Foundation of the University of Barcelona. The research groups were also supported by grants 2017SGR945 for Consolidated Research Groups, Generalitat de Catalunya, Spain. CR-C was supported by a grant from the Bosch i Gimpera Foundation (Universitat de Barcelona). The authors are members of the Institut de Recerca en Nutrició, i Seguretat Alimentària (INSA-UB), which is recognized as a Maria de Maeztu Unit of Excellence and funded by MICIN/AEI/FEDER (CEX2021-001234-M).JP is employed by APC Europe S.L.U. .The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Introduction: Natural products such as phytoestrogens-enriched foods or supplements have been considered as an alternative therapy to reduce depressive symptoms associated with menopause. It is known that the aqueous extract of Punica granatum (AE-PG) exerts antidepressant-like effects by activating β-estrogen receptors and facilitates the antidepressant response of the clinical drug citalopram (CIT). However, the effects on neuroplasticity are unknown. Objectvie investigated the antidepressant-like response of combining AE-PG and CIT at sub-optimal doses, analyzing their effects on the formation and maturation of dendrite spines in granule cells as well as on the dendrite complexity.Methods: Ovariectomized Wistar rats (3-month-old) were randomly assigned to one of the following groups: A) control  AE-PG at a sub-threshold dose (vehicle of CIT plus AE-PG at 0.125 mg/kg), C) CIT at a sub-threshold dose (0.77 mg/kg plus vehicle of AE-PG), and D) a combination of CIT plus AE-PG . All rats were treated intraperitoneally for 14 days. Antidepressant-like effects were evaluated using the force swimming test test (FST). The complexity of dendrites and the number and morphology of dendrite spines of neurons were assessed in the dentate gyrus after Golgi-Cox impregnation. The expressions of the mature brain-derived neurotrophic factor (mBDNF) in plasma and of mBDNF and synaptophysin in the hippocampus, as markers of synaptogenesis, were also determined.Results: Administration of CIT combined with AE-PG, but not alone, induced a significant antidepressant-like effect in the FST with an increase in the dendritic complexity and the number of dendritic spines in the dentate gyrus (DG) of the hippocampus, revealed by the thin and stubby categories of neurons at the granular cell layer. At the same time, an increase of mBDNF and synaptophysin expression was observed in the hippocampus of rats that received the combination of AE-PG and CIT. Estrogens are a factor in regulating emotions, mainly in women. Several studies have shown that hormonal fluctuations at some stages of women’s lives, such as the perimenopausal transition period, induce more anxiety and depressive symptoms , and thaPunica granatum (Pomegranate)  . The pomgranate) , relevangranate) , antioxigranate) , anti-migranate) , anti-cagranate) ; 2021. Agranate) .It is known that downregulation of neuroplasticity-related mechanisms, including dendritogenesis and synaptogenesis in specific brain areas, such as the hippocampus, may contribute to the pathophysiology of depression . In factThe administration of an aqueous extract of pomegranate (AE-PG) improves antidepressant-like effects of drugs of clinical use . In a prThe mBDNF is related to the dendrite spines, whose formation follows a fine-tuning process involving the sprouting of the membrane to generate filopodia, thin, stubby, and mushroom-head-type spines . InteresInstituto Nacional de Psiquiatria “Ramón de la Fuente Muñiz" (CEI-200/2015). The experimental work was developed in Mexico City  from March to April  ovariectomized (OVX) female Wistar rats, weighing 210–260 g, were used in this study. Ovariectomy was performed by ventral approximation doing an incision on the skin and muscle of female rats under anesthesia with tribromoethanol . Oviduct30 for CIT and AE-PG was calculated and dose-response curves were constructed with independent groups of rats (n = 8–10 per group) to select a non-effective dose for the combinations on FST. Doses for CIT  and AE-PG  were combined in a 1:1 proportion, and only the combination of drugs that alone did not produce behavioral effects on FST were processed for neuroplastic analysis. In all cases, AE-PG, CIT, their combination, and/or the saline solution were administered chronically for 14 days (once a day) from 9 to 10 a.m.  Control group  plus distilled water, n = 15), B) AE-PG group at a sub-threshold dose , C) CIT group at a sub-threshold dose , and D) combination of a sub-threshold dose of AE-PG plus CIT . At the end of the behavioral test, animals were euthanized by decapitation and their brain collected and prepared to analyze the formation and maturation of dendrite spines on granule cell dendrites and changes in the dendritic complexity (n = 4–5). Blood was collected to evaluate corticosterone and mBDNF (n = 4–6), and the hippocampus was dissected, divided into right and left hemisphere, and frozen at −70°C until evaluating mBDNF levels and synaptophysin protein expression (n = 4–6).3) with the floor divided into 12 equal squares (12 × 12 cm2) on which the animals could walk freely. The number of times the animal crossed the squares during a 5-min session were registered and analyzed  was dissolved in physiological saline solution (0.9% NaCl), AE-PG  was freshly prepared using distilled water. CIT and AE-PG were administered intraperitoneally (i.p.) in a volume of 4 mL/kg body weight of the animals.Corticosterone (CORT) was determined in plasma samples, and mBDNF levels were quantified from plasma and hippocampal tissue. Blood samples were obtained by decapitation and kept on ice until centrifugation  to allow plasma extraction. Plasma was kept at −80°C until analyzed with a commercial ELISA corticosterone  and BDNF immunoassay kit  according to manufacturer’s instructions. The right hemisphere containing hippocampal tissue was homogenized in lysis buffer (RIPA Lysis Buffer System: sc-24948S) with an ultrasonic apparatus. The homogenates were centrifuged at 14,000 rpm for 15 min at 4°C, and the supernatants were collected to determine the mBDNF with a commercial ELISA immunoassay kit (EMD Merck-Millipore) and total protein concentration using Bradford reagents and bovine serum albumin (BSA) as the standard. Microplates were read at 405, 450, and 595 nm in an ELISA reader .For total protein extraction, hippocampal tissue was homogenized with 300 µL of RIPA lysis buffer containing protease inhibitors . A total of 60 μg of proteins was separated by electrophoresis on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. Membranes were blocked and blots were tested using primary antibodies including synaptophysin  and Anti-GAPDH . All antibodies were incubated overnight, the next day the membrane was washed with TBST; the Goat-anti-mouse HRP secondary antibody (Advansta) was incubated for 1 h at room temperature. Membranes were washed again; images were obtained using ChemiDoc XRS+ . The relative density of the specific bands was quantified using Image Lab 6.1 software (Bio-Rad).4OH and, subsequently, dehydrated through a series of graded ethanol washes and cleared in NeoClear™  to be covered with mounting medium  and left in the dark. From each animal, 10 representative neurons, located within the granular layer of the hippocampal dentate gyrus, were selected at random using a Leica  microscope at 100X. Density and the type of dendritic spines were analyzed in 20 μm of length of the secondary dendrite of each neuron using the Reconstructor® software , their brains were removed and the mid-brain of each animal was processed for Golgi-Cox with the FD Rapid Golgi Stain TM kit according to manufacturer’s instructions of  . The tissoftware . The clasoftware .To determine whether AE-PG and CIT at sub-threshold doses were able to induce changes in the dendritic complexity of Golgi-impregnated neurons of the hippocampal dentate gyrus, the branching pattern and length of the dendritic trees of seven different Golgi-impregnated neurons per animal were evaluated by the Sholl’s concentric circles technique as previously reported . For thipost hoc test. Data from the Sholl technique were analyzed by a two-way ANOVA followed by Tukey´s post hoc test, considering the treatment and the number of dendritic branches as testing factors. In all cases, the statistical level of significance was set at p < 0.05.Results are shown as the mean ± standard error of the mean (S.E.M). Comparisons among groups were done using the Sigma Plot 12.0 software . The behavioral analysis, the hormone levels, protein expression, dendritic complexity, density, and category of dendritic spines with Golgi-Cox impregnation were analyzed with one-way analysis of variance (ANOVA) followed by Holm-Sidak 30 of AE-PG only the dose of 2 mg/kg significantly reduced the immobility behavior on FST  with a concomitant increase in climbing behavior  compared against the control group, without significant changes on swimming at any dose. CIT reduced the immobility behavior at the 12.04 mg/kg dose , increasing swimming  and climbing  in comparison to the control group  and increasing the swimming behavior  as compared against the control group. No changes were observed in climbing behavior with any combination, and no differences were observed among them.Next, three combinations of AE-PG and CIT  decreased the immobility behavior in the FST, increasing the swimming behavior (p < 0.001). Neither AE-PG, CIT, nor the combination AE-PG plus CIT modified the climbing behavior  and CIT (0.77 mg/kg) either alone or in combination were tested on the animals’ ability to cope with an acute stressful situation in the FST . ConfirmEffect of the combination of AE-PG plus CIT on dendritic complexity of granule cells in the dentate gyrus: Sholl analysis.3,237 = 26.325, p < 0.001). Here, the combination AE-PG plus CIT showed higher effects on the dendritic complexity than in the control group (p = 0.001). Similar effects were seen after the comparison with AE-PG (p < 0.001) or CIT (p < 0.001).The analysis of the dendritic complexity of Golgi-impregnated neurons in rats treated with AE-PG, CIT, or their combination  revealedpost hoc analysis of the number of intersections from soma along the dendrite showed a relevant effect of the distance  (p = 0.014) to 110 µm  compared to the control group. Two-way ANOVA revealed: interaction of treatment × distance was not significant, F3,237 = 0.560, p = 0.983. Concerning the length of the longest dendrites of hippocampal neurons, we observed no statistically significant difference among the groups (p = 0.355).In addition, the < 0.001) . The com3,16 = 11.508, p < 0.001; p = 0.002), AE-PG (p = 0.003), or CIT (p = 0.001). The analysis of the morphology of dendritic spines revealed differences in some stages of their development . However, the analysis of the categories thin . The other groups did not show differences . Finally, the treatments did not affect the latest stage of dendritic spines development identified through the mushroom morphology . Like corticosterone levels in serum, mBDNF levels were not significantly different among the different groups . However, differences among the groups were observed in hippocampal tissue  and AE-PG (p = 0.009), but not with CIT (p = 0.278).Quantification of CORT as an index of activation of the hypothalamic-pituitary-adrenal (HPA) axis revealed that chronic administration of AE-PG, CIT, and the combination AE-PG plus CIT at non-effective doses were not statistically significant different when compared among groups (p < 0.03) or its combination with CIT (p < 0.01) increased the expression of synaptophysin in samples of the hippocampus  3A family, affecting drug metabolism ; howeverIn conclusion, the results of this study support the fact that the AE-PG plus CIT combination induces antidepressant-like effects involving the increase of dendritic complexity by increasing the number of dendritic spines, primarily the thin spines category and through the participation of the hippocampal BDNF.


Lactobacillus acidophilus S-layer, contains in its C-terminus region (SlpA284 − 444), a protein domain (named here as SLAPTAG) responsible for the association of SlpA to the bacterial surface. SLAPTAG was adapted for the development of a novel affinity chromatography method: the SLAPTAG-based affinity chromatography (SAC).The S-layer proteins are a class of self-assembling proteins that form bi-dimensional lattices named S-Layer on the cell surface of bacteria and archaea. The protein SlpA, which is the major constituent of the TAG and efficiently purified by a Bacillus subtilis-derived affinity matrix (named Bio-Matrix or BM). Different binding and elution conditions were evaluated to establish an optimized protocol.Proteins with different molecular weights or biochemical functions were fused in-frame to the SLAPTAG and BM was reached after a few minutes of incubation at 4°C, with an apparent dissociation constant (KD) of 4.3μM. A reporter protein (H6-GFP-SLAPTAG) was used to compare SAC protein purification efficiency against commercial immobilized metal affinity chromatography. No differences in protein purification performance were observed between the two methods. The stability and reusability of the BM were evaluated, and it was found that the matrix remained stable for more than a year. BM could be reused up to five times without a significant loss in performance. Additionally, the recovery of bound SLAP-tagged proteins was explored using proteolysis with a SLAP-tagged version of the HRV-3c protease (SLAPASE). This released the untagged GFP while the cut SLAPTAG and the SLAPASE were retained in the BM. As an alternative, iron nanoparticles were linked to the BM, resulting in BMmag. The BMmag was successfully adapted for a magnetic SAC, a technique with potential applications in high-throughput protein production and purification.The binding equilibrium between SLAPThe SAC protocol can be adapted as a universal tool for the purification of recombinant proteins. Furthermore, the SAC protocol utilizes simple and low-cost reagents, making it suitable for in-house protein purification systems in laboratories worldwide. This enables the production of pure recombinant proteins for research, diagnosis, and the food industry. Proteins are biopolymers formed by a particular amino acid sequence that determines a given atomic spatial distribution, also known as protein conformation. Far from being a static structure, proteins behave as “nano-machines” performing precise molecular activities due to internal movements of the protein parts or protein domains Wood, . In addiProteins perform vital functions in life. For instance, some proteins break down food into nutrients during the process of digestion in the human body, while other proteins transport critical compounds for supporting life (like hemoglobin that transports oxygen to the cells), shape the cellular structure , function as hormones (like insulin), or defend the organism against pathogens (like the antibodies) Buxbaum, .In addition to the multiplicity of existent polypeptides in nature, molecular biology and biotechnology have generated a myriad of chimeric proteins by combining different protein domains Baldo, . These aHowever, for various applications such as biochemical, industrial, or medical purposes, proteins need to be purified, stabilized, and concentrated to a high degree to be useful. This condition is reached by a series of steps oriented to isolate and purify a desired protein from the other proteins in the mixture, free of contaminants, and preserving its biological activity. The purification process exploits differences in size, charge, hydrophobicity, ligand-binding affinity, or specific sequences Schales, . SeveralAffinity chromatography is a special type of liquid chromatography that exploits the existence of natural  is formulated in a specific application buffer that will favor the binding process to a ligand. Then, the sample is placed in contact with a chromatography material (or chromatography matrix) associated with the affinity ligand that will consequently recognize and retain the tagged protein. Finally, the chromatography matrix is washed several times to remove all the unbound protein. In the “OFF” phase (or elution phase), an elution buffer is passed or incubated to release the tagged protein by changing the pH or the ionic strength modifying the affinity of the interaction (non-specific elution) or by the addition of the free ligand that will out-compete the retained tagged protein (bio-specific elution)  allowed us to identify a tandem of two copies of the 60-aminoacid protein domain named SLP-A (pfam03217) which is necessary for the association  formed by the peptides EspA36 − 192, Intimin653 − 953, and Tir240 − 378 (or EIT). The resulting chimeric antigen (EIT-SLAPTAG) recombinantly expressed and purified was able to associate with the bacterial cell wall of L. acidophilus, a process that we named decoration. Thus, EIT-decorated L. acidophilus after oral administration was able to deliver the EIT antigen to the intestinal mucosa eliciting a protective immune response that controls an experimental STEC infection in mice  status, a trait that is important for vaccine purposes.Here, we explored the adaptation of a protein domain present in the carboxy terminus of the ociation . Here, tTAG for the development of novel affinity batch chromatography, the SLAP affinity chromatography (SAC), and a comprehensive protocol is presented.Here, we explore the adaptation of the SLAPEscherichia coli and Bacillus subtilis strains were grown in Luria Bertani (LB) medium  at 37°C and 180 rpm. Bacterial plasmid vectors were transformed into E. coli DH5α or E. coli BL21 (DE3) for protein expression. Pichia pastoris was grown in Yeast Extract–Peptone–Dextrose (YPD) medium at 28°C and 180 rpm. HEK293F cells were maintained at 37°C in a 5% CO2 atmosphere in Dulbecco modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum and streptomycin (50 μg/ml)–penicillin (50 U/ml). The SARS-CoV-2 Spike ectodomain Hexa-pro construction   and Rv-lys-SalI (CGGTCGACTCCACGGTCAGAAGTGACCAGTTCG). The PCR product was digested with the restriction enzymes NdeI and SalI and then cloned into the pLC3-EITH7-SlpA vector in the same restriction sites, and the recombinant plasmid was transformed into E. coli DH5α. In addition, the lysozyme gene was subcloned into pET28-e GFP-SlpA at the NdeI and NotI restriction sites and transformed into E. coli BL21 by electroporation.Lysozyme gene was amplified from the pLysS plasmid  with the oligonucleotide primers Fw-lys-EspA gene was amplified with the following primers: pRvEspAXbaI (GCTCTAGATTTACCAAGGGATATTGCTG) and pFwEITSalI (ACGCGTCGACGATATGAATGAGGCATCTAAA). After digestion with XbaI and SalI, the gene was cloned into pGEX-SlpA .The gene encoding the human rhinovirus (HRV) type 14 3C protease fused to SLAPP. pastoris GS115 transformation, 5 μg of pPICZ alpha B—hrv-3c plasmid was linearized with SacI restriction enzyme and transformed into cells through electroporation using 2 mm gap cuvettes . Transformed cells were selected by plating on a YPD medium containing Zeocin 100 ug/ml for resistance selection. Isolated colonies were further grown in test tubes containing YPD broth, and after 24 h, protein expression was induced by adding 1% (v/v) pure methanol every 24 h. The supernatants were sampled after 72 h of induction, and the best producer clones were chosen for further experiments.For BamHI and XhoI restriction enzymes, and the SLAPTAG-containing band was subcloned into SARS-CoV-2 S HexaPro plasmid (Addgene#154754) with the same restriction sites.The pET28-eGFP-SLAP was digested with E. coli BL21 were grown at 37°C and 180 rpm in liquid LB supplemented with antibiotics until they reached OD600 = 0.6. Then, 0.1 mM IPTG was added, and bacteria were grown for another 20 h at 18°C and 180 rpm. Finally, bacteria were harvested and lysed by sonication. The bacterial lysates were clarified by centrifugation.For Pichia, pastoris protein expression cells were induced with methanol. In brief, P. pastoris cultures were grown in 50 ml of YPD for 48 h until the dextrose was consumed. Then, methanol pulses (200 ul) were supplied every 24 h for 5 days. Finally, P. pastoris were harvested, and the supernatants were collected.2 for 24 h. For transfection, 4 μl of PEI was diluted in 40 μl of DMEM. 500 ng of DNA was added, and the transfection mix was incubated for 20 min at room temperature. Then, the transfection mix was transferred to the cells and incubated for 48 h. Finally, the supernatant was collected to check protein expression.HEK293 cells were transfected using polyethyleneimine (PEI) for protein expression. In brief, 30,000 cells per well were seeded in 24 well plates and incubated at 37°C, 5% COPurification of 6xHis-tagged proteins was carried out according to the manufacturer's protocol. In brief, benchtop columns were equilibrated with the binding buffer . Columns were loaded with the bacterial lysate. After being washed, columns were eluted in steps with 10-, 50-, 100-, 300-, and 500-mM imidazole in the equilibration buffer. When compared with the SAC, the IMAC batch format was adopted. Thus, Ni-NTA superflow resin (Qiagen) was equilibrated with buffer  in 1.5 ml tubes. Cleared bacterial lysates were loaded and mixed in an orbital shaker for 1 h at 4°C. The samples were centrifugated, and, after being washed, the resin was eluted with 500-mM imidazole in an equilibration buffer.B. subtilis natto was grown in 200 mL of Luria Bertani broth at 37°C and 180 rpm for 48 h. Then, the culture was centrifugated, and the bacteria were washed twice with PBS. The culture was resuspended in PBS with 2% glutaraldehyde and incubated overnight with soft agitation. Next, fixed bacteria were washed twice with PBS and stored in 20 % ethanol. 1 mL of BM correspond to DO600 = 30 of B. subtilis.BM was weighted in a drying scale (KERN MLS-D), and a calibration curve for dry weight vs. optical density was performed.To establish an optimized SAC protocol, BM was equilibrated in an initial binding buffer . Then, the samples containing SLAP-tagged proteins were incubated with the BM at different times and temperatures. After incubation, the samples were washed three times by centrifugation  using the binding buffer. Finally, as shown in the results section, elution was evaluated by incubating at different times and temperatures with different elution buffers. Based on our experimental optimization studies, we have determined that the optimal binding conditions are 200 mM NaCl and 50 mM Tris-HCl at pH 7.5 and a temperature of 4°C for 5 min. For the elution step, we found that using a carbonate-bicarbonate buffer (0.1M) with a pH of 10 and a concentration of 200 mM NaCl for 5 min was optimal.Escherichia coli, HEK293, and Pichia pastoris were purified from bacterial lysate or cellular supernatant using the final protocol described here.Once the protocol was set, proteins expressed in To analyze stability in time, aliquots of BM were frozen at −20°C. At different times, the samples were unfrozen and used for purifying GFP-SLAP following the protocol developed in this study. To analyze reusage, an aliquot of BM was used for purifying GFP-SLAP following the protocol developed in this study. After elution, the BM was washed with two volumes of the elution Buffer (0.1M carbonate-bicarbonate buffer) with a pH of 10 and a concentration of 200 mM NaCl and then two volumes of binding buffer . The cleaning process was repeated each time after elution.Protein samples were dissolved in a cracking buffer and incubated for 5 min at 100°C. Protein electrophoresis was performed at 120 V on 12% SDS-PAGE gel. Gels were stained in a Coomassie Blue solution (20% methanol and 10% acetic acid).For Western blot analysis, proteins were transferred to a nitrocellulose membrane for 55 min at 15 V using a semi-dry electroblotting transfer unit . Membranes were incubated for 1 h with blocking buffer (1% dry skim milk and 0.1% Tween in PBS). Then, membranes were incubated for 1 h with primary antibody diluted in blocking buffer (1/500). After washing with PBS-0.1% Tween, membranes were incubated for 1 h with IRDye fluorophore-labeled secondary antibodies  diluted in a blocking buffer . Finally, membranes were scanned using the Odyssey Imaging System (LI-COR).GFP fluorescence measurements were performed at 485/535 nm excitation–emission wavelength, respectively, using FilterMax F5 Microplate Reader in Black 96 Well Plates (Thermo).6)-GFP-SLAPTAG structure was predicted by AlphaFold2 -GFP-SLAPTAG on BM were performed using batch experiments. BM was equilibrated with binding buffer . Purified (H6)-GFP-SLAPTAG protein at 3 mg/ml in binding buffer was used as a stock solution. Different concentrations of protein were incubated with 10 μl of BM. After reaching equilibrium, the samples were centrifugated and GFP fluorescence from the supernatant was measured. Unbound protein in equilibrium with the BM was calculated using GFP fluorescence. Bound protein was estimated by the difference between input and unbound protein. The adsorption isotherm data were then fitted to the Langmuir isotherm equation to calculate the parameters Qmax and KD.Adsorption isotherms for . After treatment with PFA (4% in PBS), the samples were washed twice with PBS. Finally, the samples were observed with a confocal laser-scanning microscope Olympus FV1000 using a PlanApo N (60 × 1.42 NA) oil objective.TAG protein was bound to the BM. After binding, BM was washed with the same buffer thrice at 4°C. SLAPASE was added and incubated at 4°C for 60 min. Percolate containing GFP protein without SLAPTAG was recovered. As a control, the protease and the cut SLAPTAG were eluted with Bio-Matrix elution buffer after cleavage using 2M LiCl.Cleavage buffer recommended for commercial HRV3c protease was prepared: 50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol. GFP-LEVLFQGP-SLAPIron nanoparticles were synthesized by reverse co-precipitation as described by Nadi et al. . In summIn brief, 5 ml of Bio-Matrix were resuspended in PBS and iron nanoparticles were added at a final concentration of 40 g/L. The sample was mixed with gentle stirring for 30 min. Then, it was washed repeatedly with PBS 1X and preserved in 20% ethanol.TAG or GFP) using aluminum hydroxide as an adjuvant. Boosters with 5 μg of protein were further performed at 2 and 4 weeks. One week after the last immunization, the mice were bled, and the serum was stored at −20°C for later use.BALB/c mice were immunized intraperitoneally with 10 μg of the different purified recombinant proteins -GFP-SLAPTAG adapted here as a SLAPTAG reporter protein. As shown in 6)-GFP-SLAPTAG was predicted based on the AlphaFold2 method . Interestingly, although B. subtilis has no S-layer, we have demonstrated previously that this bacterium can be externally covered with SLAP-tagged proteins to generate a recombinant S-layer on its bacterial cell wall, a process that we called decoration  have the same chemical composition as Lactobacillus acidophilus. Interestingly, in B. subtilis and L. acidophilus, teichoic acid and lipoteichoic acid are distributed homogeneously on their cell wall.As a working chromatographic matrix, a culture of TAG, the reporter protein (H6)-GFP-SLAPTAG was recombinantly expressed in Escherichia coli, purified by affinity chromatography mediated by its Hisx6 tag (H6), and further incubated with BM under a variety of conditions. Initially, as shown in 6)-GFP-SLAPTAG to the BM was evaluated, finding that the best binding efficiency was reached when the incubation was performed at 4°C. To get insights into the SLAPTAG association dynamics, (H6)-GFP-SLAPTAG was incubated for 5, 30, and 60 min with BM, and as shown in 6)-GFP-SLAPTAG to BM was reached very rapidly (5 min), showing no significant increments in its association at longer time points. These results indicated that the SLAPTAG binds very rapidly and with an apparent high affinity to the BM. To quantify the affinity of the interaction between the SLAPTAG with the BM, an adsorption isotherm was performed to determine the apparent equilibrium dissociation constant as described in the Materials and Methods section. As shown in D was estimated as 4.7 μM. Interestingly, this dissociation constant value was in the same order as other KD described for different microbial S-layer proteins and their respective bacterial cell walls -GFP-SLAPTAG elution from BM, LiCl solution was selected as the positive control. As shown in 6)-GFP-SLAPTAG was achieved with high efficiency with a 2M LiCl solution at room temperature, in a short lapse of 5 min -GFP-SLAPTAG with similar efficiency as the LiCl solutions. Although CTAB was very efficient in this step, we found that this compound was difficult to remove downstream from the elution fraction. Then, taking into consideration that SLAPTAG has a theoretical isoelectric point (pI) value of 9.92, consequently, we explored whether the modification of pH can be adapted as an elution method. As shown in TAG isoelectric point (pI), (H6)-GFP-SLAPTAG was efficiently eluted from BM. In addition, after establishing the carbonate-bicarbonate buffer at pH 10 as an elution buffer, we explore whether the addition of NaCl can improve the elution process. As shown in 6)-GFP-SLAPTAG in the eluate. Interestingly, at pH 7.6, the same concentration of NaCl (present in the binding/washing buffer) has no effect on detaching SLAP-tagged proteins from BM.As it was mentioned, the SLAPof 5 min . As it w6)-GFP-SLAPTAG, the BM was fixed and immobilized on coverslips at different steps of the process of protein purification to be observed by confocal microscopy. As shown in 6)-GFP-SLAPTAG, only a red native autofluorescence was observed from fixed Bacillus subtilis natto cells present in BM. Remarkably, after the incubation of BM with (H6)-GFP-SLAPTAG, it was possible to detect the adhesion of the reporter protein to the BM establishing a recombinant fluorescent S-layer that covers completely the bacterial surface (6)-GFP-SLAPTAG was completely removed from the bacterial surface -GFP-SLAPTAG which also has a His-tag that can be purified by a metal affinity chromatography or IMAC. As described in Materials and Methods, a bacterial lysate (H6)-GFP-SLAPTAG was divided into two fractions, and both protocols were performed accordingly in parallel, confirming that SAC optimized protocol was able to purify (H6)-GFP-SLAPTAG with high efficiency -GFP-SLAPTAG being the BM stable for more than a year that was tested (14 months). In addition, BM reusability capacity was evaluated determining that the matrix can be reused five times with no modification of the protein purification yield , recombinantly expressed, and purified (ASE activity was generated (GFP-LEVLFQGP-SLAPTAG)  , and a p6)-GFP-SLAPTAG was purified in a few and easy steps. As shown in the SDS-PAGE (mag) was able to purify the reporter protein (H6)-GFP-SLAPTAG very efficiently  . In B. subtilis-derived matrix, named here as Bio-Matrix (BM), showed a high performance in its binding and elution capacities, combined with good purification parameters for our reporter protein (H6)-GFP-SLAPTAG. The results presented here show that the SAC protocol can be potentially adapted as a universal tool for recombinant protein purification. Thus, proteins from different origins, with different molecular weights, or produced by different recombinant expression systems  can be efficiently purified by SAC. In addition to its universal application, we demonstrate here that SAC was able to achieve a protein yield similar to those obtained by commercial affinity chromatography systems such as the immobilized metal affinity chromatography or IMAC. As reported, in protein production bioprocess, chromatography is the most expensive step  are naturally integrated into the surface of the BM . Additionally, we investigated different potential chromatography materials that can directly bind SLAPandidate .One outstanding aspect of SAC was its ability to efficiently capture SLAP-tagged proteins from a high-concentration protein solution, such as an unclarified crude sample  . ImportaPichia pastoris supernatant, or HEK293 cell supernatant in a single step , the enzymatic cleavage of the tag is still preferred as it is the most controlled process, with no premature cleaving and the best yields are obtained  was approved by the Committee on the Ethics of Animal Experiments of the Universidad Nacional de San Martín (UNSAM), under the recommendations for animal experimentation .Pichia pastoris experiments. MR contribute in the manuscript writing and results discussion of the manuscript. GB is the P.I. and director of the project, contributing to experiment design, result discussion, and writing of the manuscript. All authors contributed to the article and approved the submitted version.EM performed most of experimental work and also generate figures and contributed to discussion of manuscript. PU performed molecular cloning of multiples proteins used. GE perform experiments regarding mammalian cells proteins expression. MP perform microscopy results. CD perform